Zhang Chunbin, Xiong Qingming, Kikuchi Takane, Rikihisa Yasuko
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210-1093, USA.
Clin Vaccine Immunol. 2008 Mar;15(3):402-11. doi: 10.1128/CVI.00366-07. Epub 2007 Dec 19.
Ehrlichia ewingii, a tick-transmitted rickettsia previously known only as a canine pathogen, was recently recognized as a human pathogen. E. ewingii has yet to be cultivated, and there is no serologic test available to diagnose E. ewingii infection. Previously, a fragment (505 bp) of a single E. ewingii gene homologous to 1 of 22 genes encoding Ehrlichia chaffeensis immunodominant major outer membrane proteins 1 (OMP-1s)/P28s was identified. The purposes of the present study were to (i) determine the E. ewingii omp-1 gene family, (ii) determine each OMP-1-specific peptide, and (iii) analyze all OMP-1 synthesized peptides for antigenicity. Using nested touchdown PCR and a primer walking strategy, we found 19 omp-1 paralogs in E. ewingii. These genes are arranged in tandem downstream of tr1 and upstream of secA in a 24-kb genomic region. Predicted molecular masses of the 19 mature E. ewingii OMP-1s range from 25.1 to 31.3 kDa, with isoelectric points of 5.03 to 9.80. Based on comparative sequence analyses among OMP-1s from E. ewingii and three other Ehrlichia spp., each E. ewingii OMP-1 oligopeptide that was predicted to be antigenic, bacterial surface exposed, unique in comparison to the other E. ewingii OMP-1s, and distinct from those of other Ehrlichia spp. was synthesized for use in an enzyme-linked immunosorbent assay. Plasmas from experimentally E. ewingii-infected dogs reacted significantly with most of the OMP-1-specific peptides, indicating that multiple OMP-1s were expressed and immunogenic in infected dogs. The results support the utility of the tailored OMP-1 peptides as E. ewingii serologic test antigens.
尤因埃立克体，一种以前仅作为犬类病原体为人所知的蜱传播立克次体，最近被确认为人类病原体。尤因埃立克体尚未培养成功，且尚无用于诊断尤因埃立克体感染的血清学检测方法。此前，已鉴定出尤因埃立克体单个基因的一个片段（505 bp），该片段与编码恰菲埃立克体免疫显性主要外膜蛋白1（OMP-1s）/P28s的22个基因中的1个同源。本研究的目的是：（i）确定尤因埃立克体omp-1基因家族；（ii）确定每种OMP-1特异性肽段；（iii）分析所有合成的OMP-1肽段的抗原性。我们采用巢式降落PCR和引物步移策略，在尤因埃立克体中发现了19个omp-1旁系同源基因。这些基因在一个24 kb的基因组区域中，串联排列在tr1下游和secA上游。预测的19种成熟尤因埃立克体OMP-1的分子量范围为25.1至31.3 kDa，等电点为5.03至9.80。基于尤因埃立克体与其他三种埃立克体属的OMP-1之间的比较序列分析，合成了每种预测具有抗原性、暴露于细菌表面、与其他尤因埃立克体OMP-1相比独特且与其他埃立克体属不同的尤因埃立克体OMP-1寡肽，用于酶联免疫吸附测定。实验感染尤因埃立克体的犬的血浆与大多数OMP-1特异性肽段有显著反应，表明多种OMP-1在感染犬中表达且具有免疫原性。这些结果支持定制的OMP-1肽作为尤因埃立克体血清学检测抗原的实用性。
